Identification of the NRF2 transcriptional network as a therapeutic target for trigeminal neuropathic pain

Trigeminal neuralgia, historically dubbed the “suicide disease,” is an exceedingly painful neurologic condition characterized by sudden episodes of intense facial pain. Unfortunately, the only U.S. Food and Drug Administration (FDA)–approved medication for trigeminal neuralgia carries substantial side effects, with many patients requiring surgery. Here, we identify the NRF2 transcriptional network as a potential therapeutic target. We report that cerebrospinal fluid from patients with trigeminal neuralgia accumulates reactive oxygen species, several of which directly activate the pain-transducing channel TRPA1. Similar to our patient cohort, a mouse model of trigeminal neuropathic pain also exhibits notable oxidative stress. We discover that stimulating the NRF2 antioxidant transcriptional network is as analgesic as inhibiting TRPA1, in part by reversing the underlying oxidative stress. Using a transcriptome-guided drug discovery strategy, we identify two NRF2 network modulators as potential treatments. One of these candidates, exemestane, is already FDA-approved and may thus be a promising alternative treatment for trigeminal neuropathic pain.


INTRODUCTION
Trigeminal neuralgia is a chronic, debilitating neuropathy characterized by sudden, short, and intense episodes of shooting, stabbing, or shock-like pain in the face (1)(2)(3). The pain can be triggered by activities of everyday life, such as eating, drinking, talking, or brushing teeth. For some patients, even a simple breeze across their face can trigger excruciating pain (4,5). This pain is so debilitating that trigeminal neuralgia was historically dubbed the "suicide disease" because patients would sometimes take their own life to end their suffering (6). Many more bear the pain but endure a poor quality of life, anxiety, and depression (7).
Typical trigeminal neuralgia is thought to result from vascular compression of the trigeminal nerve, the principal sensory nerve of the face (8,9). This compression can injure and demyelinate the nerve, rendering it hyperexcitable and prone to generating ectopic action potentials that may then be interpreted as pain (10,11).
Unfortunately, the current medical treatments for trigeminal neuralgia fall short. The only U.S. Food and Drug Administration (FDA)-approved drug for managing trigeminal neuralgia is the anticonvulsant carbamazepine, which broadly and nonspecifically inhibits neural activity (12). Carbamazepine also carries a substantive side effect profile, including hyponatremia and life-threatening drug reactions (13)(14)(15). Patients who fail medical management may resort to surgery, in which microsurgical dissection frees the nerve from the offending artery, or the compressive vein is cauterized and divided (16). Microvascular decompression is often effective, with 61 to 80% of patients reporting sustained pain relief years after surgery (17). However, this still leaves some patients who experience persistent or recurrent pain. Evidence from long-term follow-up suggests that despite maximum medical and surgical/percutaneous interventions, many patients encounter pain recurrence and incomplete pain control, representing an unmet clinical need (18).
To date, there remains an incomplete understanding of the pathophysiologic molecular mechanisms underlying trigeminal neuralgia. Approximately 25% of patients with trigeminal neuralgia do not exhibit vascular compression of the nerve from the outset (3,18,19). About half of these cases may be attributed to secondary causes such as multiple sclerosis or neoplasms, both of which are thought to demyelinate and injure the trigeminal nerve (20)(21)(22). In the other half, the underlying cause remains unknown (19,23).
These different etiologies appear to ultimately converge upon nerve injury itself. A common consequence of neural injury and inflammation is the generation of reactive oxygen species (ROS), a class of redox-active small molecules (24). When uncontrolled, ROS dysregulate cellular processes by inappropriately oxidizing and modifying biomolecules, leading to lipid peroxidation, protein oxidation, and DNA damage (25,26). Aberrant ROS signaling may contribute to neuropathic pain as well (27)(28)(29). Several ROS, including hydrogen peroxide (H 2 O 2 ) and hypochlorite (OCl − ), can directly activate the pain-transducing channel transient receptor potential ankyrin 1 (TRPA1) (30,31). In several animal models of sciatic nerve neuropathic pain, antagonizing ROS by administering antioxidants systemically or intrathecally reduces hyperalgesia and relieves allodynia (27,32). However, the sciatic and trigeminal nerves diverge functionally and transcriptionally in response to painful stimuli, with injury to trigeminal neurons being more intense and more difficult to treat (33,34).
Here, we report evidence of increased oxidative stress contributing to trigeminal neuropathic pain. A mouse model of trigeminal neuralgia similarly accumulates ROS, several of which directly activate TRPA1. Consistent with previous work describing TRPA1 as a nociceptor in animals (29,35), we find that pharmacologically inhibiting or genetically eliminating TRPA1 blunts pain. However, attempts at TRPA1 inhibition have been challenging to translate clinically, with multiple failed trials (36,37). We discover that stimulating the nuclear factor erythroid 2-related factor 2 (NRF2) antioxidant transcriptional network is as powerfully analgesic as directly inhibiting TRPA1 while also reversing underlying oxidative stress. Using a transcriptome-guided drug discovery approach, we identify two NRF2 network modulators as potential treatments. One of these candidates, exemestane, is an FDA-approved drug used to treat estrogen receptor-positive breast cancer and could be readily repurposed as a mechanistically different treatment for trigeminal neuropathic pain.

Patients and a mouse model of trigeminal neuralgia exhibit increased oxidative stress
Microvascular decompression requires a craniotomy, presenting a unique opportunity to sample cerebrospinal fluid (CSF) from patients with trigeminal neuralgia. We evaluated patients' CSF for evidence of oxidative stress by measuring 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), two major products of lipid peroxidation (38,39). Compared to CSF collected from patients undergoing posterior fossa craniectomies (to release Chiari malformations), shunts (to relieve normal pressure hydrocephalus or pseudotumor cerebri), or lumbar punctures (to ease pseudotumor cerebri), both 4-HNE and MDA are elevated in CSF from patients with trigeminal neuralgia (Fig. 1, A to C). CSF 4-HNE and MDA do not correlate with CSF hemoglobin, suggesting that they are not blood contaminants during microvascular decompression (fig. S1, A and B). 4-HNE and MDA are unlikely to be artifacts of surgery or anesthesia either, since both are still elevated in patients with trigeminal neuralgia compared to controls who underwent similar posterior fossa craniectomies (fig. S1, C and D). Instead, the accumulation of CSF 4-HNE and MDA in trigeminal neuralgia may reflect elevated oxidative stress.
To monitor and manipulate the influence of oxidative stress in trigeminal neuralgia, we use a mouse model throughout this study. In this model, branches of the trigeminal nerve are chronically constricted with a loose ligature. As previously reported by several groups (40,41), constricting branches of the trigeminal nerve in mice elicits allodynia and hyperalgesia in the region innervated by the damaged nerve (Fig. 1D).
Constricting the maxillary division of trigeminal nerve in mice elicits both mechanical and cold allodynia (Fig. 1, E and F). Mice exhibit heightened nocifensive behavior to crude touch with a von Frey filament (Fig. 1E) and to the application of ice-cold acetone to the affected vibrissal pad skin surface (Fig. 1F). Similar to our patient cohort, this mouse model also exhibits notable oxidative stress. Ligating the maxillary nerve results in increased levels of 4-HNE (Fig. 1G), protein carbonylation (Fig. 1H), and MDA ( Fig. 1I) in nerve lysates, mirroring the oxidative stress in CSF from patients with trigeminal neuralgia.

TRPA1 is activated by ROS and mediates trigeminal neuropathic pain
As ROS accumulate in the constrictive mouse model of trigeminal neuralgia, one mechanism by which they may elicit pain is by activating the pain-transducing channel TRPA1 (30,31). TRPA1 is a nonselective cation channel located at the plasma membrane of both pain-and itch-encoding sensory neurons (42)(43)(44). TRPA1 not only is a principal sensor of noxious cold (35,42,45,46) but also is capable of sensing environmental irritants and reactive molecules such as iodoacetamide (43,(47)(48)(49) ROS directly activate TRPA1 by covalently bonding or modifying a network of cysteine and lysine residues within the channel. Extensive work by several groups has pinpointed a collection of key residues in TRPA1, including Cys 421 , Cys 621 , Cys 641 , Cys 665 , and Lys 721 (49,51,52). To test which residues among these sense H 2 O 2 and 4-HNE, we mutated each individually and in various combinations ( fig. S2, A and B). Triply mutating cysteines Cys 621 , Cys 641 , and Cys 665 to serine renders TRPA1 insensitive to both iodoacetamide and H 2 O 2 (Fig. 2, D and E) (51). As previously described, this triple mutant is still activated by 4-HNE but is no longer sensitive after mutating nearby Lys 721 (Fig. 2F). These four residues position TRPA1 as both a key sensor and transducer of oxidative stress.
Since TRPA1 senses H 2 O 2 and 4-HNE in isolation, we wondered whether it may respond similarly to CSF from patients with trigeminal neuralgia. Of the 13 trigeminal neuralgia cases we screened, CSF from all but 4 activates TRPA1-expressing HEK cells. In contrast, CSF from 9 of 11 control patients does not activate TRPA1expressing cells (P = 0.0188) (fig. S3, A to P). Moreover, CSF from patients with trigeminal neuralgia activates cells expressing TRPA1 but not cells transfected with the vector backbone alone ( Fig. 2G and  fig. S3, I to P), suggesting that components of the CSF specifically activate TRPA1. CSF from each patient with trigeminal neuralgia activates more than 20% of iodoacetamide-responsive cells, whereas CSF from control patients activates less than 2% of iodoacetamideresponsive cells (P = 0.0031) (Fig. 2H). To confirm that CSF from patients with trigeminal neuralgia can also activate TRPA1 in its native, neuronal environment, we applied CSF from patients with trigeminal neuralgia and controls to wild-type (WT) and TRPA1-null (TRPA1 −/− ) trigeminal neuronal cultures. To distinguish pain-and itch-encoding neurons from other neurons in both WT and TRPA1 −/− cultures, we filtered our analysis to neurons that also responded to the transient receptor potential cation channel subfamily V member 1 (TRPV1) agonist capsaicin (53). Previous studies have demonstrated that 97% of TRPA1-positive sensory neurons express TRPV1, and thus, TRPV1 can serve to unbiasedly identify pain-and itch-encoding sensory neurons in the absence of TRPA1 expression (42,48). By this definition, CSF from patients with trigeminal neuralgia activates almost 40% of WT capsaicin-responsive neurons but only 11% of TRPA1 −/− neurons (P = 0.0016) (Fig. 2, I and J). CSF from control patients does not activate any sampled WT neurons, further suggesting that unique components of CSF from patients with trigeminal neuralgia can activate endogenous TRPA1.
As endogenous TRPA1 responds to CSF from patients, we tested whether pharmacologically inhibiting or genetically eliminating TRPA1 would blunt pain in the constrictive mouse model of trigeminal neuralgia. We find that WT mice treated with the TRPA1 inhibitor AM-0902 (54) exhibit reduced mechanical and cold allodynia (Fig. 2, K and L). Mice that genetically lack TRPA1 exhibit less mechanical and cold allodynia as well, consistent with recent work implicating TRPA1 as a major mediator of trigeminal neuropathic pain in mice (Fig. 2, M and N) (29). Both TRPA1 −/− males and females exhibit less mechanical and thermal allodynia ( fig. S4, A to D).

Activating NRF2 attenuates trigeminal neuropathic pain and oxidative stress
The potent antinocifensive effects of blocking or eliminating TRPA1 suggest that its inhibition might be a therapeutic strategy in managing trigeminal neuralgia. TRPA1 inhibitors are being actively considered in clinical trials for postoperative pain and diabetic neuropathy (55). Unfortunately, several trials have already been abandoned because of disappointing pharmacokinetics or poor efficacy (36,37). We considered whether we could instead prevent TRPA1 activation by dampening the underlying damaging oxidative stress.
A principal cellular defense mechanism against oxidative or electrophilic stress is activation of the NRF2 antioxidant response One day after habituation, mice underwent constriction of the maxillary nerve or a sham surgery. From the next day onward until day 10, mice were evaluated every day for mechanical allodynia. On day 11, mice were evaluated for cold hypersensitivity. Mice were habituated for 30 min before behavior testing every day. (E) Scored mechanical allodynia and (F) timed cold allodynia from mice that underwent constriction of the maxillary nerve or sham surgery. Points in (E) represent the means ± SEM of n = 5 (sham) and 10 (constriction). Points in (F) represent individual mice. (G and H) Immunoblots and analysis of (G) 4-HNE and (H) protein carbonylation from maxillary nerves of mice that underwent constriction or sham surgery, normalized to -actin. Lanes and points represent individual mice. (I) Quantification of MDA from maxillary nerves of mice that underwent constriction or sham surgery normalized to protein (micrograms of MDA per milligram of protein). Points represent individual mice. (B, C, and F to I) Median and range depicted. *P < 0.05 and **P < 0.01 by two-tailed unpaired Student's t test. (56,57). NRF2 (or Nfe2l2) is a ubiquitously expressed transcription factor that governs the expression of a network of antioxidant genes, including Nqo1, Gsta2, and Hmox1 (58-60). As a master regulator of the cellular redox state, NRF2 is tightly regulated. NRF2 is constitutively expressed but, under basal conditions, is continually tagged for proteasomal degradation by the E3-ubiquitin ligase KEAP1 (61,62). Similar to TRPA1, KEAP1 harbors several redox-sensitive cysteines that are readily modified by electrophiles and oxidants (63)(64)(65). Oxidation of these cysteines by ROS inhibits KEAP1 (66,67), stabilizing NRF2 such that it can then translocate to the nucleus to induce the expression of antioxidant and cytoprotective genes. As TRPA1 seems central to pain in the constrictive mouse model of trigeminal neuralgia, we wondered whether activating the NRF2 antioxidant network might lessen allodynia by countering redox stress. Mice treated with the KEAP1 inhibitor sulforaphane (68) are less sensitive to both crude touch and cold than vehicle-treated mice after constricting the maxillary nerve (Fig. 3, B and C). However, sulforaphane did not lower mechanical or cold allodynia in TRPA1 −/− mice any further compared with genetic deletion of TRPA1 alone, suggesting that oxidative stress contributes to pain upstream of, and possibly through, TRPA1 ( fig. S5, A and B). Pretreating mice with sulforaphane limits oxidative stress after nerve constriction and reduces the levels of 4-HNE (Fig. 3D), protein carbonylation (Fig. 3E), and MDA (Fig. 3F). By immunohistochemistry, we find that sulforaphane increases NRF2 expression in neurons of the trigeminal ganglion (Fig. 3G).
Whereas lowering redox stress by activating NRF2 is analgesic, we also considered whether raising redox stress by deleting NRF2 (NRF2 −/− ) can lead to hyperalgesia or allodynia. Consistent with such a model, we found that NRF2 −/− mice exhibit greater mechanical and cold allodynia after constriction of the maxillary nerve compared to WT mice (Fig. 3H). Somewhat analogously, Yang et al. (69) have reported that NRF2 −/− mice are hypersensitive to oxaliplatininduced peripheral neuropathy. Just as with WT mice, however, treating NRF2 −/− mice with the TRPA1 antagonist AM-0902 lowers both mechanical and cold allodynia, suggesting that oxidative stress is epistatic to TRPA1, with TRPA1 transducing the increased oxidative stress into hyperalgesia and allodynia (Fig. 3, I and J). NRF2 is also activated by lower concentrations of 4-HNE and H 2 O 2 than TRPA1, suggesting that TRPA1 is activated during periods of relatively greater oxidative stress. Specifically, we find that 4-HNE stimulates NRF2 at a median effective concentration (EC 50  Although sulforaphane activates NRF2, it can exert additional confounding pharmacologic activities (70,71). Therefore, we genetically augmented the NRF2 transcriptional network by eliminating KEAP1 altogether. We generated Keap1-floxed [Keap1(f/f)] mice that contain a tamoxifen-inducible Cre recombinase (Fig. 4, A and B). Fibroblasts isolated from these mice were treated with vehicle or 4-hydroxytamoxifen (4-OHT) and subsequently genotyped to confirm that Keap1 is retained unless inducibly targeted and excised (Fig. 4, C and D). When treated with tamoxifen, mice harboring both Cre recombinase and floxed Keap1 alleles exhibit loss of Keap1 with a concomitant increase in Nqo1, a canonical NRF2 target gene (Fig. 4, E and F). Compared to mice lacking Cre recombinase, mice harboring both Cre and floxed Keap1 alleles exhibit significantly less mechanical and cold allodynia after nerve constriction (Fig. 4, H and I). Eliminating Keap1 generally matches the response we observe with sulforaphane, although the analgesic effect of eliminating Keap1 is slightly more consistent across days than with sulforaphane (Figs. 3B and 4H).

Drug repositioning identifies NRF2 network modulators as potential treatments for trigeminal neuropathic pain
If limiting oxidative stress can lessen allodynia in patients as it does in mice, then inducing the NRF2 transcriptional network may be an alternative approach to managing trigeminal neuralgia. Unfortunately, sulforaphane exhibits somewhat poor pharmacokinetics that limit its utility in humans (72,73). To screen for candidate alternatives to sulforaphane, we adapted a transcriptome-guided drug discovery scheme termed transcriptome reversal (74,75). Transcriptome reversal posits that if a dysregulated transcriptome drives a particular disease, then correcting the transcriptome back toward a normal state may be therapeutic. To reverse the dysregulated transcriptome, the pathologic genetic signature is compared to the transcriptomes of cells treated with different small molecules. Molecules with transcriptome signatures that anticorrelate with the disease signature are prioritized for further validation. The Connectivity Map (CMAP) (74,76) provides publicly available expression signatures derived from cell lines treated with thousands of small molecules. Transcriptomic approaches that have leveraged CMAP and other resources have successfully identified targeted therapeutics for cancers (77), as well as diabetes, inflammatory bowel disease, and neurodevelopmental disorders (75,78,79).
To identify therapeutic candidates that induce the NRF2 transcriptional network, we queried compounds that best mimic the transcriptional signature of overexpressing Nfe2l2 and genetically silencing Keap1 (Figs. 3A and 4G and table S1). CMAP scores how well each compound's transcriptional profile matches the query signature from −100 to +100, with a score of +100 indicating a complete match. In the Nfe2l2-queried signature, 187 compounds received a Connectivity Score above CMAP's recommended cutoff of +90, whereas 114 compounds scored above +90 in the Keap1-derived signature (Fig. 5, A and B, and table S1). Among the top 20 candidate compounds prioritized per signature, 7 compounds overlapped between the two queries (Fig. 5C). We focused on exemestane and JQ-1, two overlapping candidate compounds with high scores in both the Nfe2l2-derived and Keap1-derived signatures. Exemestane is an FDA-approved aromatase inhibitor indicated for the treatment of estrogen receptor-positive breast cancer (80,81). JQ-1 is an inhibitor of the BET family of bromodomain-containing proteins, displacing them from acetylated lysine residues on histones (82). BET inhibitors structurally similar to JQ-1 are being considered in clinical trials to treat a variety of cancers (83).
To test whether exemestane or JQ-1 induces the NRF2 transcriptional network as predicted in silico, we applied both compounds to a reporter cell line in which changes in NRF2 activity are coupled to the expression of firefly luciferase. In these cells, the promoter controlling luciferase expression contains several NRF2 binding sites, thereby directly tying luciferase expression to NRF2 activity. As expected, inhibiting KEAP1 with sulforaphane dose-dependently increases luciferase expression in the NRF2 reporter line. Exemestane similarly increases luciferase expression (Fig. 5D) and up-regulates the canonical NRF2 target Nqo1 (84), suggesting that exemestane also promotes NRF2 transcriptional activity. In contrast, JQ-1 does not stimulate luciferase expression, ostensibly suggesting that it does not induce the NRF2 transcriptional network (Fig. 5D). However, subsequent reanalysis of our published RNA sequencing data (85) uncovered that JQ-1 unexpectedly up-regulates a number of canonical NRF2 target genes in primary human dermal fibroblasts, such as NQO1, FTL, PRDX1, TXN, and EGR1 ( Fig. 5E and table S2). JQ-1 also up-regulates Nqo1 and Hmox1 in mouse corneal fibroblasts and monocytes (86,87). Unbiased gene ontology and genetic network analyses detected that the NRF2 pathway is the second most up-regulated pathway after treatment with JQ-1 ( Fig. 5F and table S3). JQ-1 thus induces much of the NRF2 transcriptional network, but by a mechanism that may not involve classical NRF2 signaling. Consistent with this, we find that JQ-1 neither stabilizes NRF2 nor inhibits its ubiquitination, whereas both sulforaphane and exemestane do. Both sulforaphane and exemestane also promote nuclear translocation of NRF2, but JQ-1 does not (Fig. 6, A to D). JQ-1 might instead up-regulate antioxidant genes by remodeling chromatin through BET proteins, stimulating NRF2 in an unconventional manner (86,88), or activating alternative transcription factors (89), but the exact mechanisms remain presently unclear.
As both exemestane and JQ-1 recruit the NRF2 transcriptome, we evaluated whether they could lower allodynia in the constrictive mouse model of trigeminal neuralgia. Just as with sulforaphane, mice treated with either exemestane or JQ-1 are much less sensitive to both crude touch (Fig. 7, A and C) and the application of ice-cold acetone to the ligated vibrissal pad skin surface (Fig. 7, B and D). By immunohistochemistry, we find that exemestane robustly increases NRF2 expression in neurons of the trigeminal ganglion (Fig. 7E). Directly applying exemestane to the maxillary nerve similarly lessens both mechanical and cold allodynia (Fig. 7, F and G), suggesting that exemestane may also exert analgesia locally at the nerve.
To assess whether exemestane or JQ-1 acts through alternative off-target mechanisms, we also evaluated nocifensive behavior in WT mice treated with either letrozole or (−)-JQ-1. Similar to exemestane, letrozole is a potent aromatase inhibitor but is structurally unrelated (90) and does not induce Nqo1 (84). (−)-JQ-1 is the inactive stereoisomer of (+)-JQ-1 (Fig. 8, A and B) (82). We observe that while exemestane and (+)-JQ-1 lower both mechanical and cold allodynia, neither letrozole nor (−)-JQ-1 does (Fig. 8, C to F). The analgesic activity of exemestane and JQ-1 is thus less likely due to alternative effects from aromatase inhibition or nonspecific changes in DNA topology. To next test whether sulforaphane, exemestane, and JQ-1's analgesic activities are dependent on NRF2, we treated NRF2 −/− mice with all three drugs. We find that both sulforaphane and exemestane lose their analgesic effects in NRF2  To determine a ratio of KEAP1 to NRF2 cDNA that best mimics baseline NRF2 activity, luciferase activity was measured 48 hours after transfection with varying ratios of KEAP1 to NRF2 cDNA, normalized to total protein. Luciferase expression is controlled by a promoter that contains several NRF2 binding sites and is thus a measure of NRF2 activity. A 2.5 g of KEAP1-to-1 g of NRF2 cDNA ratio returned luciferase activity to baseline and was therefore used for subsequent biochemical studies evaluating NRF2 stability and nuclear translocation. that JQ-1 does not biochemically influence NRF2 in the same manner as sulforaphane and exemestane (Fig. 6), this further suggests that how JQ-1 up-regulates the NRF2 transcriptional network may only partially depend on NRF2 itself. Whether JQ-1 stimulates other transcription factors or chromatin remodeling (89) will require future investigation.
Neither sulforaphane, exemestane, nor JQ-1 inhibits TRPA1 channel activity, suggesting that their analgesic effects are not mediated by directly inhibiting the nociceptor itself ( fig. S8, A to C). Sulforaphane, exemestane, and JQ-1 do not affect TRPA1 expression either ( fig. S9, A to H). Instead, exemestane and JQ-1 may limit oxidative stress. Similar to sulforaphane, pretreating mice with exemestane or JQ-1 reduces the levels of 4-HNE ( fig. S10, A and C) and protein carbonylation ( fig. S10, B and D) after nerve constriction.
We considered whether bypassing NRF2 and directly administering an antioxidant might also reduce allodynia. Several groups have reported that antioxidants can lower hyperalgesia and allodynia in other animal models of neuropathic pain like sciatic chronic constriction injury (27) and spinal nerve constriction (91), but the effect is notably acute and only lasts a few hours. In contrast, the effects of sulforaphane, exemestane, and JQ-1 persist 24 hours after treatment. We find that treating mice with the antioxidant ascorbate does not reduce either mechanical or cold allodynia (fig. S11, A and B) or the levels of 4-HNE and protein carbonylation (fig. S11, C and D) at 24 hours, underscoring the value of specifically targeting the NRF2 anti oxidant network in sustaining analgesia. Because most small-molecule antioxidants such as ascorbate act by stoichiometrically scavenging ROS directly, they are likely most effective when administered very frequently and at high dosages.

DISCUSSION
Trigeminal neuralgia is a chronic, debilitatingly painful condition. Unfortunately, medical treatments for trigeminal neuralgia often fall short, in part because the pathophysiology is incompletely understood. Currently, the only FDA-approved drug for managing trigeminal neuralgia is the anticonvulsant carbamazepine, which broadly and nonspecifically inhibits neural activity (12,14). However, carbamazepine carries a notable side effect profile, including hyponatremia, leukopenia, ataxia, and the risks of drug reaction with eosinophilia and systemic symptoms and Stevens-Johnson syndromes (13)(14)(15). Other drugs with fewer side effects such as gabapentin, pregabalin, and antidepressants are sometimes prescribed off-label but are less effective than carbamazepine (17). Up to 20 to 40% of patients with trigeminal neuralgia are also prescribed opiates for their pain despite little evidence supporting their use (92,93), potentially worsening the socioeconomic and medical burdens of opiate overdose and addiction. Alternative treatments such as radiofrequency and/or glycerin rhizotomy or stereotactic radiosurgery use heat, chemicals, or radiation to ablate the trigeminal nerve to blunt pain. However, these procedures can leave patients with postprocedural numbness or devastating anesthesia dolorosa. In light of these limitations, we sought to better understand the pathophysiology underlying trigeminal neuralgia.
By leveraging a combined clinical, molecular, and computational approach, the present study identifies the NRF2 transcriptional network as a potential therapeutic target for trigeminal neuropathic pain (Fig. 9). Using a transcriptome-guided drug discovery approach, we identify exemestane and JQ-1 as two candidate NRF2 network modulators for treating trigeminal neuropathic pain. We find that exemestane induces the NRF2 network through NRF2 itself, whereas JQ-1 recruits the network differently. In contrast to current pharmacologic agents that mask pain by blunting nerve firing, increasing the NRF2 transcriptional network may be a therapeutic approach that seeks to improve pain through redox control.
A few patients on systemic exemestane report musculoskeletal stiffness. Recently, Fusi et al. (94) rigorously examined whether exemestane can elicit these symptoms through TRPA1. They find that exemestane can elicit acute allodynia in mice but within 3 hours of dosing. We observe that at the dose of 10 mg/kg used in many preclinical models (95,96), exemestane exerts a powerful analgesic effect that persists over the course of days (Fig. 7, A and B). If normalizing dose to body surface area through allometric scaling, a mouse dose of 10 mg/kg is equivalent to 0.8 mg/kg in humans or 48 mg for a 60-kg person. This dose is well within the 25-to 50mg dosing used clinically. In addition, the maximum serum concentration that exemestane reaches in patients falls between 0.05 and 0.1 M (97, 98), 500-to 1000-fold lower than the EC 50 value at which exemestane activates TRPA1 in vitro. Thus, we propose that clinically relevant concentrations of exemestane may not activate TRPA1 but that supratherapeutic concentrations might and thus should be avoided when treating patients ( fig. S12, A and B). We also note that directly applying exemestane to the trigeminal nerve is also analgesic in mice (Fig. 7, F and G), suggesting that percutaneous, stereotactic administration of exemestane to the trigeminal nerve in humans may be a safe and feasible approach to treating trigeminal neuralgia while minimizing the potential adverse effects of systemic exemestane (94). Further drug discovery could alternatively refine exemestane or identify new exemestane-like agents to minimize potential adverse effects. In contrast, neither sulforaphane nor JQ-1 activates TRPA1 ( fig. S12, C and D).
While our findings suggest that elevated oxidative stress contributes to trigeminal neuropathic pain in both patients and mice, they do not exclude other mechanisms. Recently, Trevisan et al. (29) found that depleting monocytes and macrophages with clodronate or an antibody against the chemoattractant chemokine ligand 2 lessens allodynia in a mouse model of trigeminal neuralgia. Their findings demonstrate that inflammation contributes to trigeminal neuralgia by eliciting redox stress. In this same vein, the Kultima group found that inflammatory biomarkers are elevated in the CSF from patients with trigeminal neuralgia (99,100). Paracrine signaling by TRPA1 in non-neural cells may also promote neuroinflammation (101). In addition, TRPA1 may be recruited to the membrane (102) or acutely sensitized (103) during episodes of pain. Neuronally, aberrant ephaptic coupling (104) and afferent circuit plasticity (105) may also be interpreted as pain.
A final note is that the preclinical model used in this and other studies involves constriction of the maxillary segment of the trigeminal nerve, which is a location distinct from the cisternal segment that is compressed by the superior cerebellar artery in most patients with typical trigeminal neuralgia. An improved model would involve the intradural compression of the preganglionic trigeminal nerve, although the feasibility of this study in mice is questionable. Whether this commonly used preclinical model sheds mechanistic insight into typical trigeminal neuralgia remains to be fully proven (106). Nevertheless, our data reveal that targeting the NRF2 transcriptional network alone is analgesic, suggesting that repurposing NRF2 transcriptional network modulators may be a mechanistically alternative approach toward managing trigeminal neuropathic pain.

Experimental model and subject details Human
Institutional review board approval. Patients with trigeminal neuralgia, Chiari malformations, idiopathic normal pressure hydrocephalus, or pseudotumor cerebri were recruited under protocols approved by the Institutional Review Board at the Johns Hopkins University School of Medicine (study numbers IRB00103861 and NA_00029413).
Cerebrospinal fluid. CSF from patients was collected into specimen tubes, transferred to 15-ml conical tubes, and subsequently centrifuged for 5 min at 2000g within 2 hours of collection. The supernatant was then collected, aliquoted, and stored at −80°C until experimentation. Mice Animal care and use. All experiments were performed in accordance with protocols approved by the Animal Care and Use Committee at the Johns Hopkins University School of Medicine.
Keap1(f/f) mice. Keap1(f/f) mice were generated as previously described (107). Tamoxifen-inducible Keap1(f/f)/CMV-CreER mice were generated by crossing Keap1(f/f) mice with CAG-CreER + mice. To induce excision of Keap1, Keap1(f/f)/CMV-CreER was injected intraperitoneally with tamoxifen (75 mg/kg) once every 24 hours over five consecutive days. Keap1(f/f) (Cre-negative) mice were similarly injected with tamoxifen and served as controls (Fig. 4C). Disruption of Keap1 was confirmed by genomic polymerase chain reaction (PCR) using primers listed in Table 1 (Key resources table) and by quantitative RNA PCR for Keap1 (Fig. 4, D and E). Nrf2 activation was confirmed by measuring the expression of the canonical target NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) quinone oxidoreductase 1 (Nqo1) by quantitative RNA PCR (Fig. 4F). Behavioral and molecular tests were only performed seven or more days after the final tamoxifen injection. Patients and a mouse model of trigeminal neuropathic pain exhibit increased oxidative stress, as evidenced through elevated levels of ROS (red). As ROS accumulate, one mechanism by which they may elicit pain is by activating the pain-transducing channel TRPA1 (green). ROS directly activate TRPA1 by covalently bonding or modifying a network of cysteine and lysine residues within the channel (Cys). The transcription factor NRF2 (gray) is a master regulator of the cellular redox state, governing the expression of a network of antioxidant genes. Recruiting the NRF2 transcriptome pharmacologically with the small molecules exemestane or JQ-1 lowers oxidative stress, thereby alleviating mechanical and cold allodynia in the mouse model of trigeminal neuropathic pain.   NRF2 knockout (NRF2 −/− ) mice. NRF2 −/− mice were generated as previously described (108) and purchased from the Jackson Laboratory (stock no. 017009).

Method details Materials and preparation
Compounds. Compounds were obtained as follows: sulforaphane Material preparation. All drugs were freshly prepared in an appropriate solvent immediately before beginning each treatment course. Each drug was then aliquoted into individual tubes for each day and stored at −20°C before thawing at 4°C just before treatment. Sulforaphane was dissolved in 100% dimethyl sulfoxide (DMSO) and then diluted to 1% DMSO in saline. Exemestane, letrozole, (+)-JQ-1, and (−)-JQ-1 were dissolved in 100% DMSO and then diluted to 5% DMSO in corn oil. Ascorbate was dissolved directly in saline. AM-0902 was dissolved in 100% DMSO and then diluted to 5% DMSO in olive oil. Tamoxifen was dissolved directly in corn oil. All other compounds were prepared as 50-to 1000-l aliquots and stored at −20°C before thawing at 4°C. Freeze/thaw cycles were avoided whenever possible.

Constriction of the maxillary nerve
Constriction of the maxillary division of the trigeminal nerve was performed as previously described (40,41). The procedure was performed under direct visualization and control with a surgical microscope. Mice were first anesthetized with ketamine [100 mg/kg, intraperitoneally (i.p.)] and xylazine (12.5 mg/kg, i.p.) and monitored by pinching the skin between the toes with forceps and monitoring for withdrawal. Mice were restrained with adhesive tape to a sterilized polystyrene board. Upon sufficient anesthesia, the scalp was shaved, and an anterior-to-posterior skin incision was made at the midline to expose the nasal and maxillary bones. The maxillary nerve was exposed and carefully dissected free from the surrounding connective tissue. The distal end of the maxillary nerve was then loosely constricted using 8-0 silk sutures as a ligature. The sutures were tied using a slip knot followed by a normal knot, after which any remaining suture was cut free. The incision was then closed with a 4-0 silk suture. In the sham procedure, the left maxillary nerve was exposed but not constricted. Mice were monitored and rehydrated until fully recovered from anesthesia.

Pain behavior assays
Mechanical allodynia. Mechanical allodynia was assessed in C57BL/6J, TRPA1 +/+ , TRPA1 −/− , Keap1(f/f), and Keap1(f/f)/CMV-CreER mice using a Von Frey filament as previously outlined (40,41). Animals were placed individually in transparent plastic boxes and allowed to acclimate to the environment for at least 30 min before testing. After habituation, a 0.04-g force von Frey filament was used to stimulate the territory innervated by the maxillary nerve, including the vibrissal skin pad. Each mouse's response to the filament was scored from 0 to 4 on the scale below. Each mouse was scored 10 times per day. Changes in mechanical allodynia were considered relative to sham-or vehicle-treated animal controls. Behavior was assessed concomitantly or in a blocked manner with consideration for both genotype and treatment as follows: 0, no response; 1, nondefensive response to the stimulus (i.e., mouse nondefensively explores the filament); 2, withdrawal response (i.e., mouse turns its head away from the filament); 3, escape/attack response (i.e., mouse moves its body away from the filament and assumes crouching position against the box wall; actively attacks the filament by biting and/or grabbing); 4, asymmetric face grooming (i.e., mouse wipes the stimulated facial area in an uninterrupted series of at least three face-wash strokes). Cold allodynia. Cold allodynia was assessed in C57BL/6J, TRPA1 +/+ , TRPA1 −/− , Keap1(f/f), and Keap1(f/f)/CMV-CreER mice using icecold acetone as previously outlined (109). Animals were placed individually in transparent plastic boxes and allowed to acclimatize to the environment for at least 30 min before testing. After habituation, 20 l of cold acetone was applied to the ligated vibrissal pad skin surface. Cold allodynia was measured as the average time spent wiping the region in a 60-s period, allowing for a lull of 5 s between bouts of wiping. Allodynia was measured three times with 10 min between intervals. Changes in cold allodynia were considered relative to shamor vehicle-treated animal controls. Behavior was assessed concomitantly or in a blocked manner with consideration for both genotype and treatment. Immunoblotting Western blotting. Western blotting was performed as previously described (110,111). Briefly, tissues were homogenized at 4°C in lysis buffer [solution of 50 mM tris-HCl, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, and 1% Triton X-100 (pH 7.4)] supplemented with protease inhibitors (Sigma-Aldrich). When dissecting maxillary nerves for immunoblot analysis, nerves were dissected both as proximally toward the semilunar ganglion and as distal to the skin as possible. Nerves were harvested 11 days after surgery, immediately after the final behavioral test. Lysates were then pulse-sonicated and centrifuged at 16,000g for 10 min at 4°C. Fifteen micrograms of cleared lysate was run on a 4 to 12% polyacrylamide Bis-Tris gradient gel in running buffer [solution of 50 mM MES, 50 mM tris base, 0.1% SDS, and 1 mM EDTA (pH 7.3)] and then transferred to a polyvinylidene difluoride membrane. Membranes were blocked with 5% milk in TBS-T [solution of 16 mM tris-HCl, 140 mM NaCl, and 0.1% Tween 20 (pH 7.6)] for 1 hour at 25°C and then incubated with primary antibodies in 3% bovine serum albumin (BSA) (w/v) in TBS-T overnight at 4°C. The following day, membranes were washed with TBS-T and then incubated with secondary antibodies in 3% BSA (w/v) in TBS-T for 1 hour at 25°C. The following primary antibodies were used: rabbit anti-4-HNE (1:1000; Abcam, ab46545), rabbit anti-TRPA1 (1:1000; Novus Biologicals, NB110-40763), rabbit anti-2,4dinitrophenol (1:150; Millipore Sigma, 90451), and mouse anti--actin [1:10,000; Santa Cruz Biotechnology, sc-47778 horseradish peroxidase (HRP)]. The following secondary antibodies were used: donkey anti-rabbit immunoglobulin G (IgG) (1:10,000; GE Healthcare, NA934) and goat anti-rabbit (1:300; Millipore Sigma, 90452). Immunoblots were quantified by densitometry as described below in the "Quantification and statistical analysis" section.
Dot blotting. Ten microliters of each CSF sample was spotted onto a nitrocellulose membrane in a dot blot manifold and vacuumdried. Membranes were then blocked with 3% BSA (w/v) in TBS-T for 1 hour at 25°C and then incubated with a rabbit anti-4-HNE (1:1000; Abcam, ab46545) primary antibody in 3% BSA (w/v) overnight at 4°C. The following day, membranes were washed with TBS-T and then incubated with a donkey anti-rabbit IgG (1:10,000; GE Healthcare, NA934) secondary antibody in 3% BSA for 1 hour at 25°C. Oxidative stress assays 4-Hydroxynonenal. 4-HNE was quantified by immunoblot as described above in the "Western blotting" section with a rabbit anti-4-HNE (1:1000; Abcam, ab46545) primary antibody.
Protein carbonylation. Protein carbonylation was assessed with the OxyBlot Protein Oxidation Detection Kit (listed in Table 1) as per the manufacturer's instructions. Protein carbonylation was quantified by immunoblot as described above in the "Western blotting" section with a rabbit anti-DNP (1:150; Millipore Sigma, 90451) primary antibody.
Malondialdehyde. MDA was quantified with the OxiSelect TBARS Assay Kit (listed in Table 1) as per the manufacturer's instructions. Concentrations below the limit of detection (LOD) were censored and substituted with a constant value of the LOD/√2 (112). Hemoglobin ELISA CSF hemoglobin was quantified with the Human Hemoglobin ELISA (enzyme-linked immunosorbent assay) Kit (listed in Table 1) as per the manufacturer's instructions. Concentrations below the LOD were censored and substituted with a constant value of the LOD/√2 (112). Cell culture and transfection HEK-293 cells. HEK-293 cells were cultured in DMEM, 10% fetal bovine serum, penicillin/streptomycin (100 U/ml), and glutamine (2 mM) in an atmosphere of 5% CO 2 at 37°C as previously described (111).
Transfection. Transfection was performed as previously described (111). HEK-293 cells were transiently transfected with Lipofectamine 3000 (Thermo Fisher Scientific) transfection reagents according to the manufacturer's instructions. Cells were harvested 24 to 48 hours after transfection for downstream analysis. Immunoprecipitation Immunopreciptation was performed as previously described with a few modifications (111). Briefly, cells were harvested on ice in lysis buffer [50 mM tris-HCl, 150 mM NaCl, 1% Triton X-100, and 5% glycerol (v/v) (pH 7.6 solution)] supplemented with protease inhibitors and centrifuged at 16,000g for 10 min at 4°C. Anti-c-Myc affinity gel (Millipore Sigma) was added to cleared lysates and rotated overnight at 4°C. Five percent of each sample was set aside as input before addition of beads. The following day, beads were pelleted by centrifugation at 1000g for 30 s at 4°C. The supernatant was aspirated, and beads were washed five times in lysis buffer by repeat centrifugation, aspiration, and resuspension. Proteins were eluted from beads by boiling for 5 min in lithium dodecyl sulfate sample buffer (Thermo Fisher Scientific), and the eluates were analyzed by immunoblot as described above. Subcellular fractionation Subcellular (nuclear versus cytoplasmic) fractionation was performed as previously described with a few modifications (113). Briefly, HEK-293 cells transiently transfected with the proteins of interest were lysed and snap-frozen in subcellular lysis buffer [10 mM Hepes, 1 mM EGTA, 1 mM dithiothreitol, and 10% sucrose (w/v) (pH 7.4 solution)] supplemented with protease inhibitors. The lysates were than thawed and filtered through a 70-m nylon filter to remove intact cells. An aliquot of the thawed filtered lysate was retained as the whole-cell fraction, and the remaining lysate was centrifuged at 1000g for 10 min at 4°C. The resulting supernatant was retained as the crude membrane/cytoplasmic fraction, and the pellet was washed two times in lysis buffer by repeat resuspension, centrifugation, aspiration, and resuspension. The pellet was then resuspended in lysis buffer and retained as the nuclear fraction. The membrane/ cytoplasmic fraction was then centrifuged at 21,000g for 20 min at 4°C, and the resulting supernatant was retained as the cytoplasmic fraction. The whole-cell, cytoplasmic, and nuclear fractions were then sonicated on ice and clarified one last time at 16,000g for 10 min at 4°C. The fractions were then analyzed by immunoblot as described above.

Calcium imaging and analysis
Calcium imaging and analysis were performed as previously described (114 HEK-293 cells. HEK-293 cells were plated on poly-d-lysine-coated coverslips and transiently transfected with the vector backbone or constructs encoding WT or mutant TRPA1. Unless otherwise noted, cells were imaged for 30 s to establish a baseline before compounds were added. Vehicle was first applied for 30 s, after which 100 M iodoacetamide, 1 mM H 2 O 2 , or 100 M 4-HNE was applied. CSF from cases and controls was diluted into CIB 1:50 before each trial. Fifty micromolar of the nonselective TRP channel inhibitor ruthenium red was applied at the end of every imaging trial. Trigeminal ganglia. Neurons were incubated with Fluo-4 AM 12 to 16 hours after dissociation. Unless otherwise noted, neurons were imaged for 30 s to establish a baseline before compounds were added. Vehicle was first applied for 30 s, after which CSF was applied for 60 s and then 100 nM capsaicin for 30 s. Randomly pooled CSF from cases or controls was diluted into CIB 1:1 before each trial. At the end of every imaging trial, 50 mM KCl was added as a positive control. Percentage activated was determined as described earlier. To distinguish pain-and itch-encoding neurons from other neurons in the culture, we filtered our analysis to neurons that also responded to the TRPV1 agonist capsaicin (53). Previous studies have demonstrated that TRPA1 and TRPV1 are coexpressed in pain-and itch-encoding sensory neurons, and either can serve to unbiasedly identify such neurons (42,48). NRF2/ARE luciferase reporter assay NRF2 activity was monitored with a reporter cell line in which changes in NRF2 activity are coupled to the expression of firefly luciferase (NRF2/ARE Luciferase Reporter HEK-293 Stable Cell Line, listed in Table 1). Luciferase activity was quantified with the Luciferase Assay System (listed in Table 1) as per the manufacturer's instructions. Briefly, cells were plated at 125,000 cells per well in a 24-well plate. The following day, cells were treated with either vehicle or varying doses of sulforaphane, exemestane, or JQ-1 for 6 hours. Cells were then lysed in passive lysis buffer (Promega, E1941) supplemented with protease inhibitors (Sigma-Aldrich). Lysates were then cleared at 16,000g for 15 min at 4°C. Fifteen microliters of each lysate was mixed with 5 l of luciferase assay substrate (Promega). Total light was measured using a luminometer with a 10-s integration time with a delay of 2 s. Luciferase measurements were normalized to total protein and reported as normalized to the vehicle condition. Histology Maxillary nerves from mice were dissected and fixed in cold 4% formaldehyde (v/v) overnight at 4°C. Tissues were then cryoprotected through a series of 10, 20, and 30% sucrose (w/v) gradients for 24 hours each at 4°C. Tissues were then embedded in Optimal Cutting Temperature compound and sectioned in 20-m intervals with a cryostat, after which the sections were dried onto slides and kept at −20°C. Sections were then processed for immunohistochemistry.

RNA sequencing and analysis
Sequencing. RNA sequencing was performed as previously described (85). To assess the transcriptional signature of JQ-1, total RNA was isolated from three independent human fibroblast lines after 48 hours of treatment with DMSO or 0.25 M JQ-1 using TRIzol and RNeasy isolation columns (Qiagen) as per the manufacturer's instructions. DNA was digested with deoxyribonuclease treatment. All samples had RNA integrity numbers 9.60 or higher as measured with an Agilent 2100 Bioanalyzer. mRNA was enriched by polyadenylate selection, prepped using an Illumina TruSeq mRNA sample preparation kit, and sequenced by Illumina HiSeq 2000.
Differential gene expression and pathway enrichment analyses. Differential gene expression analysis was performed as previously described (111) using BioJupies (115). We leveraged BioJupies to perform Pathway Enrichment Analyses through Enrichr (116) to identify the biological processes that are overrepresented in the gene set up-regulated by JQ-1 treatment.

DNA/RNA isolation, PCR, and quantitative PCR
Total cellular or tissue DNA/RNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen) for DNA or the RNeasy Plus Universal Kit (Qiagen) for RNA as per the manufacturer's instructions, as previously described (111). PCR was performed with the Platinum Taq DNA Polymerase High Fidelity Kit (Invitrogen), whereas qPCR was performed with the TaqMan RNA-to-Ct 1-Step Kit (Applied Biosystems). All reagents are listed in Table 1.

In silico candidate drug screening
We queried the CMAP (clue.io) to identify molecules most likely to reproduce the transcriptional signature of both overexpressing Nfe2l2 (the gene encoding NRF2) and genetically silencing Keap1. CMAP scores how well each compound's transcriptional profile matches the query signature from −100 to +100, with a score of +100 indicating a complete match. Molecules with higher scores were interpreted as most likely mimicking up-regulating the NRF2 transcriptional network. In the Nfe2l2-queried signature, 187 compounds receive a Connectivity Score above CMAP's recommended cutoff of +90, whereas 114 compounds score above +90 in the Keap1-derived signature. Exemestane and JQ-1 were two overlapping candidate compounds with high scores in both the Nfe2l2-derived and Keap1-derived signatures and were thus prioritized for further validation. Exemestane scored 99.5772 in the Nfe2l2-derived signature and 95.587 in the Keap1-derived signature. JQ-1 scored 99.3935 in the Nfe2l2-derived signature and 99.7503 in the Keap1-derived signature.

Quantification and statistical analysis
Quantification and statistical analysis were performed as previously described (111). All data were plotted and expressed as the median and range, means ± SEM, or means ± 95% CI, as noted. Statistical comparisons were performed using two-tailed unpaired Student's t tests, Fisher's exact tests, or analysis of variance (ANOVA) analyses, as noted with tests correcting for multiple testing as appropriate. Differences were considered significant at P < 0.05.
For densitometry analyses of immunoblots, background was subtracted with a rolling ball radius of 50.0 pixels and/or by measuring signal at an empty segment of the membrane. Protein carbonylation levels were quantified by averaging the densitometry of each lane from the highest to lowest molecular weights at which signal was 10 U above background and then normalizing to -actin. 4-HNE levels were quantified by summating densitometries of individual regions within lanes at which signal was 10 U above background and then normalizing to -actin.
All in vivo experiments were performed concomitantly or in a blocked manner with consideration for both genotype and treatment. Mice were excluded from behavioral analysis if they displayed signs of vehicle or drug toxicity or severe, unrelenting pain.

SUPPLEMENTARY MATERIALS
Supplementary material for this article is available at https://science.org/doi/10.1126/ sciadv.abo5633 View/request a protocol for this paper from Bio-protocol.